GoFish: A versatile nested PCR strategy for environmental DNA assays for marine vertebrates

Here we describe GoFish, a strategy for single-species environmental DNA (eDNA) presence/absence assays using nested PCR. The assays amplify a mitochondrial 12S rDNA segment with vertebrate metabarcoding primers, followed by nested PCR with M13-tailed, species-specific primers. Sanger sequencing confirms positives detected by gel electrophoresis. We first obtained 12S sequences from 77 fish specimens for 36 northwestern Atlantic taxa not well documented in GenBank. Using these and existing 12S records, we designed GoFish assays for 11 bony fish species common in the lower Hudson River estuary and tested seasonal abundance and habitat preference at two sites. Additional assays detected nine cartilaginous fish species and a marine mammal, bottlenose dolphin, in southern New York Bight. GoFish sensitivity was equivalent to Illumina MiSeq metabarcoding. Unlike quantitative PCR (qPCR), GoFish does not require tissues of target and related species for assay development and a basic thermal cycler is sufficient. Unlike Illumina metabarcoding, indexing and batching samples are unnecessary and advanced bioinformatics expertise is not needed. From water collection to Sanger sequencing results, the assay can be carried out in three days. The main limitations to this approach, which employs metabarcoding primers, are the same as for metabarcoding, namely, inability to distinguish species with shared target sequences and inconsistent amplification of rarer eDNA. In addition, the performance of the 20 assays reported here as compared to other single-species eDNA assays is not known. This approach will be a useful addition to current eDNA methods when analyzing presence/absence of known species, when turnaround time is important, and in educational settings

Molecular species identification of Central European ground beetles (Coleoptera: Carabidae) using nuclear rDNA expansion segments and DNA barcodes

<p>Abstract</p> <p>Background</p> <p>The identification of vast numbers of unknown organisms using DNA sequences becomes more and more important in ecological and biodiversity studies. In this context, a fragment of the mitochondrial cytochrome <it>c </it>oxidase I (COI) gene has been proposed as standard DNA barcoding marker for the identification of organisms. Limitations of the COI barcoding approach can arise from its single-locus identification system, the effect of introgression events, incomplete lineage sorting, numts, heteroplasmy and maternal inheritance of intracellular endosymbionts. Consequently, the analysis of a supplementary nuclear marker system could be advantageous.</p> <p>Results</p> <p>We tested the effectiveness of the COI barcoding region and of three nuclear ribosomal expansion segments in discriminating ground beetles of Central Europe, a diverse and well-studied invertebrate taxon. As nuclear markers we determined the 18S rDNA: V4, 18S rDNA: V7 and 28S rDNA: D3 expansion segments for 344 specimens of 75 species. Seventy-three species (97%) of the analysed species could be accurately identified using COI, while the combined approach of all three nuclear markers provided resolution among 71 (95%) of the studied Carabidae.</p> <p>Conclusion</p> <p>Our results confirm that the analysed nuclear ribosomal expansion segments in combination constitute a valuable and efficient supplement for classical DNA barcoding to avoid potential pitfalls when only mitochondrial data are being used. We also demonstrate the high potential of COI barcodes for the identification of even closely related carabid species.</p

Identification of Birds through DNA Barcodes

Short DNA sequences from a standardized region of the genome provide a DNA barcode for identifying species. Compiling a public library of DNA barcodes linked to named specimens could provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing. Recent work suggests that sequence diversity in a 648-bp region of the mitochondrial gene, cytochrome c oxidase I (COI), might serve as a DNA barcode for the identification of animal species. This study tested the effectiveness of a COI barcode in discriminating bird species, one of the largest and best-studied vertebrate groups. We determined COI barcodes for 260 species of North American birds and found that distinguishing species was generally straightforward. All species had a different COI barcode(s), and the differences between closely related species were, on average, 18 times higher than the differences within species. Our results identified four probable new species of North American birds, suggesting that a global survey will lead to the recognition of many additional bird species. The finding of large COI sequence differences between, as compared to small differences within, species confirms the effectiveness of COI barcodes for the identification of bird species. This result plus those from other groups of animals imply that a standard screening threshold of sequence difference (10× average intraspecific difference) could speed the discovery of new animal species. The growing evidence for the effectiveness of DNA barcodes as a basis for species identification supports an international exercise that has recently begun to assemble a comprehensive library of COI sequences linked to named specimens

Structural Analysis of Biodiversity

Large, recently-available genomic databases cover a wide range of life forms, suggesting opportunity for insights into genetic structure of biodiversity. In this study we refine our recently-described technique using indicator vectors to analyze and visualize nucleotide sequences. The indicator vector approach generates correlation matrices, dubbed Klee diagrams, which represent a novel way of assembling and viewing large genomic datasets. To explore its potential utility, here we apply the improved algorithm to a collection of almost 17000 DNA barcode sequences covering 12 widely-separated animal taxa, demonstrating that indicator vectors for classification gave correct assignment in all 11000 test cases. Indicator vector analysis revealed discontinuities corresponding to species- and higher-level taxonomic divisions, suggesting an efficient approach to classification of organisms from poorly-studied groups. As compared to standard distance metrics, indicator vectors preserve diagnostic character probabilities, enable automated classification of test sequences, and generate high-information density single-page displays. These results support application of indicator vectors for comparative analysis of large nucleotide data sets and raise prospect of gaining insight into broad-scale patterns in the genetic structure of biodiversity

jMOTU and Taxonerator: Turning DNA Barcode Sequences into Annotated Operational Taxonomic Units

BACKGROUND: DNA barcoding and other DNA sequence-based techniques for investigating and estimating biodiversity require explicit methods for associating individual sequences with taxa, as it is at the taxon level that biodiversity is assessed. For many projects, the bioinformatic analyses required pose problems for laboratories whose prime expertise is not in bioinformatics. User-friendly tools are required for both clustering sequences into molecular operational taxonomic units (MOTU) and for associating these MOTU with known organismal taxonomies. RESULTS: Here we present jMOTU, a Java program for the analysis of DNA barcode datasets that uses an explicit, determinate algorithm to define MOTU. We demonstrate its usefulness for both individual specimen-based Sanger sequencing surveys and bulk-environment metagenetic surveys using long-read next-generation sequencing data. jMOTU is driven through a graphical user interface, and can analyse tens of thousands of sequences in a short time on a desktop computer. A companion program, Taxonerator, that adds traditional taxonomic annotation to MOTU, is also presented. Clustering and taxonomic annotation data are stored in a relational database, and are thus amenable to subsequent data mining and web presentation. CONCLUSIONS: jMOTU efficiently and robustly identifies the molecular taxa present in survey datasets, and Taxonerator decorates the MOTU with putative identifications. jMOTU and Taxonerator are freely available from http://www.nematodes.org/

DNA barcoding works in practice but not in (neutral) theory.

BACKGROUND: DNA barcode differences within animal species are usually much less than differences among species, making it generally straightforward to match unknowns to a reference library. Here we aim to better understand the evolutionary mechanisms underlying this usual "barcode gap" pattern. We employ avian barcode libraries to test a central prediction of neutral theory, namely, intraspecific variation equals 2 Nµ, where N is population size and µ is mutations per site per generation. Birds are uniquely suited for this task: they have the best-known species limits, are well represented in barcode libraries, and, most critically, are the only large group with documented census population sizes. In addition, we ask if mitochondrial molecular clock measurements conform to neutral theory prediction of clock rate equals µ. RESULTS: Intraspecific COI barcode variation was uniformly low regardless of census population size (n = 142 species in 15 families). Apparent outliers reflected lumping of reproductively isolated populations or hybrid lineages. Re-analysis of a published survey of cytochrome b variation in diverse birds (n = 93 species in 39 families) further confirmed uniformly low intraspecific variation. Hybridization/gene flow among species/populations was the main limitation to DNA barcode identification. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first large study of animal mitochondrial diversity using actual census population sizes and the first to test outliers for population structure. Our finding of universally low intraspecific variation contradicts a central prediction of neutral theory and is not readily accounted for by commonly proposed ad hoc modifications. We argue that the weight of evidence-low intraspecific variation and the molecular clock-indicates neutral evolution plays a minor role in mitochondrial sequence evolution. As an alternate paradigm consistent with empirical data, we propose extreme purifying selection, including at synonymous sites, limits variation within species and continuous adaptive selection drives the molecular clock

A Scalable Method for Analysis and Display of DNA Sequences

Background: Comparative DNA sequence analysis provides insight into evolution and helps construct a natural classification reflecting the Tree of Life. The growing numbers of organisms represented in DNA databases challenge treebuilding techniques and the vertical hierarchical classification may obscure relationships among some groups. Approaches that can incorporate sequence data from large numbers of taxa and enable visualization of affinities across groups are desirable. Methodology/Principal Findings: Toward this end, we developed a procedure for extracting diagnostic patterns in the form of indicator vectors from DNA sequences of taxonomic groups. In the present instance the indicator vectors were derived from mitochondrial cytochrome c oxidase I (COI) sequences of those groups and further analyzed on this basis. In the first example, indicator vectors for birds, fish, and butterflies were constructed from a training set of COI sequences, then correlations with test sequences not used to construct the indicator vector were determined. In all cases, correlation with the indicator vector correctly assigned test sequences to their proper group. In the second example, this approach was explored at the species level within the bird grouping; this also gave correct assignment, suggesting the possibility of automated procedures for classification at various taxonomic levels. A false-color matrix of vector correlations displayed affinities among species consistent with higher-order taxonomy

12S gene metabarcoding with DNA standard quantifies marine bony fish environmental DNA, identifies threshold for reproducible detection, and overcomes distortion due to amplification of non‐fish DNA

Abstract Single‐species PCR assays accurately measure eDNA concentration. Here we test whether multi‐species PCR, that is, metabarcoding, with an internal standard can quantify eDNA of marine bony fish. Replicate amplifications with Riaz 12 S gene primers were spiked with known amounts of a non‐fish vertebrate DNA standard, indexed separately, and sequenced on an Illumina MiSeq. Fish eDNA copies were calculated by comparing fish and standard reads. Relative reads were directly proportional to relative DNA copies, with average and maximum variance between replicates of about 1.3‐ and 2‐fold, respectively. There was an apparent minimum threshold for consistent amplification of about 10 eDNA copies per PCR reaction. The internal DNA standard corrected for suppression of fish read counts due to amplification of non‐fish DNA. To assess potential PCR bias among species, we compared reads obtained with Riaz 12 S primers to those with modified MiFish primers, which target a different 12 S gene segment. Our results provide evidence that Riaz 12 S gene metabarcoding with an internal DNA standard quantifies marine bony fish eDNA over a range of about 10–5000 copies per reaction, without indication of significant PCR bias among teleost species. In mid‐Atlantic coastal samples, eDNA rarity was the main limitation to reproducible detection and quantification, and this was partly overcome by increasing the amount of a DNA sample amplified. Our findings support incorporating a DNA standard in 12 S metabarcoding to help quantify eDNA abundance for marine bony fish species